FabK variant

ABSTRACT

The invention provides FabK polypeptides and polynucleotides encoding FabK polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are preferred methods for utilizing FabK polypeptides and polynucleotides as diagnostic reagents and in diagnostic assays to screen for microbial infections in organisms and infestations in materials.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/968,123, filed onOct. 1, 2001, now abandoned, which claims priority to U.S. ProvisionalApplication No. 60/238,503, filed on Oct. 6, 2000, both of which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,and their uses. In particular, the invention relates to polynucleotidesand polypeptides of the FabK family, as well as their variants, hereinreferred to as “FabK,” “FabK polynucleotide(s),” and “FabKpolypeptide(s),” as the case may be.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

Infections caused by or related to Streptococcus pneumoniae are a majorcause of human illness worldwide, and the frequency of resistance tostandard antibiotics has risen dramatically over the last decade. Hence,there exists an unmet medical need for diagnostic tests and reagents forthis organism.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the FabK embodiments of the invention that have a present benefit of,among other things, being useful to as diagnostic reagents. Suchreagents are also useful to determine the frequency and geographicalrange of microbial strains.

A FabK enzyme, involved in fatty acid biosynthesis, has been recentlyreported from a strain of Streptococcus pneumoniae (Heath, et al. Nature406: 145 (2000)). The present invention provides a variant form of FabK,differing at Thr 318.

The specific activity of the known enzyme under the published conditionswas 64+/−4 nmol min⁻¹, too low to efficiently perform certain biologicalanalyses of the enzyme, such as detailed enzymological analyses (Heath,et al. Nature 406: 145 (2000)). The present invention solves thisproblem by providing a variant of the known FabK described, as well asmethod useful to perform detailed enzymological studies of FabK, whereinFabK activity is sufficient to perform such studies.

Moreover, the present invention provides a method of a diagnosingbacterial infection and bacterial genotyping using FabK polynucleotidesand polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to FabK, in particular FabK polypeptidesand FabK polynucleotides, recombinant materials and methods for theirproduction. In another aspect, the invention relates to diagnosticassays and reagents for detecting diseases associated with microbialinfections and conditions associated with such infections, such asassays and reagents for detecting FabK expression or activity, or thepresence of Streptococcus pneumoniae in a host or material.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to FabK polypeptides and polynucleotides asdescribed in greater detail elsewhere herein. In particular, theinvention relates to polypeptides and polynucleotides of a FabK ofStreptococcus pneumoniae. The invention relates especially to FabKhaving a nucleotide and amino acid sequences set out in Table 1 as SEQID NO:1 and SEQ ID NO:2 respectively. Note that sequences recited in theSequence Listing below as “DNA” represent an exemplification of theinvention, since those of ordinary skill will recognize that suchsequences can be usefully employed in polynucleotides in general,including ribopolynucleotides.

TABLE 1 FabK Polynucleotide and Polypeptide Sequences (A) Streptococcuspneumoniae FabK polynucleotide sequence. [SEQ ID NO:1]5′-ATGAAAACGCGTATTACAGAATTATTGAAGATTGACTATCCTATTTTCCAAGGAGGGATGGCCTGGGTTGCTGATGGTGATTTGGCAGGGGCTGTTTCCAAGGCTGGAGGATTAGGAATTATCGGTGGGGGAAATGCCCCGAAAGAAGTTGTCAAGGCCAATATTGATAAAATCAAATCATTGACTGATAAACCCTTTGGGGTCAACATCATGCTCTTATCTCCCTTTGTGGAAGATATCGTGGATCTCGTTATTGAAGAAGGTGTTAAAGTTGTCACAACAGGAGCAGGAAATCCAAGCAAGTATATGGAACGTTTCCATGAAGCTGGGATAATCGTTATTCCTGTTGTTCCTAGTGTCGCTTTAGCTAAACGCATGGAAAAAATCGGTGCAGACGCTGTTATTGCAGAAGGAATGGAAGCTGGGGGGCATATCGGTAAATTAACAACCATGACCTTGGTGCGACAGGTACCCACAGCTATATCTATTCCTGTTATTGCTGCACGAGGAATTGCGGATGGTGAAGGTGCTGCGGCTGGCTTTATGCTAGGTGCAGAGGCTGTACAGGTGGGGACACGGTTTGTAGTTGCAAAAGAGTCGAATGCCCATCCAAACTACAAGGAGAAAATTTTAAAAGCAAGGGATATTGATACTACGATTTCAGCTCAGCACTTTGGTCATGCTGTTCGTGCTATTAAAAATCAGTTGACTAGAGATTTTGAACTGGCTGAAAAAGATGCCTTTAAGCAGGAAGATCCTGATTTAGAAATCTTTGAACAAATGGGAGCAGGTGCCCTAGCCAAAGCAGTTGTTCACGGTGATGTGGATGGTGGCTCTGTCATGGCAGGTCAAATCGCAGGGCTTGTTTCTAAAGAAGAAACAGCTGAAGAAATCCTAAAAGATTTGTATTACGGAGCCGCTAAGAAAATTCAAGAAGAAGCCTCTCGCTGGGCAGGAGTTGTAAGAAATGACTAA-3′

(B) Streptococcus pneumoniae FabK polypeptide sequence deduced from apolynucleotide sequence in this table. [SEQ ID NO:2]NH₂-MKTRITELLKIDYPTFQGGMAWVADGDLAGAVSKAGGLGIIGGGNAPKEVVKANIDKIKSLTDKPFGVNIMLLSPFVEDIVDLVIEEGVKVVTTGAGNPSKYMERFHEAGIIVIPVVPSVALAKRMEKIGADAVIAEGMEAGGHIGKLTTMTLVRQVATAISIPVIAAGGIADGEGAAAGFMLGAEAVQVGTRFVVAKESNAHPNYKEKILKARDIDTTISAQHFGHAVRAIKNQLTRDFELAEKDAFKQEDPDLEIFEQMGAGALAKAVVIGDVDGGSVAAGQIAGLVSKEETAEEILKDLYYGAAKKIQEEASRWAGVVRND-COOH

Deposited Materials

A deposit comprising a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on 11, Apr. 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit. On 17,Apr. 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli wassimilarly deposited with the NCIMB and assigned deposit number 40800.The Streptococcus pneumoniae strain deposit is referred to herein as“the deposited strain” or as “the DNA of the deposited strain.”

The deposited strain comprises a full length FabK gene. The sequence ofthe polynucleotides comprised in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is comprisedin the deposited strain. Further provided by the invention are FabKpolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare FabK polypeptide and polynucleotide sequences isolated from thedeposited strain.

Polypeptides

FabK polypeptide of the invention is substantially phylogeneticallyrelated to other proteins of the FabK family.

In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as “FabK” and “FabKpolypeptides” as well as biologically, diagnostically and clinicallyuseful variants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention arevariants of FabK polypeptide encoded by naturally occurring alleles of aFabK gene. The present invention further provides for an isolatedpolypeptide that: (a) comprises or consists of an amino acid sequencethat has at least 95% identity, most preferably at least 97–99% or exactidentity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2;(b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence that has at least 95% identity,even more preferably at least 97–99% or exact identity to SEQ ID NO:1over the entire length of SEQ ID NO:1; (c) a polypeptide encoded by anisolated polynucleotide comprising or consisting of a polynucleotidesequence encoding a polypeptide that has at least 95% identity, evenmore preferably at least 97–99% or exact identity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular a mature polypeptide) as well as polypeptidesand fragments, particularly those that are useful as diagnosticreagents, as well as those that have at least 95% identity to apolypeptide of Table 1 [SEQ ID NO:2]. Also included by the invention areportions of such polypeptides generally comprising at least 30 aminoacids, and more preferably comprising at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:X—(R₁)_(m)—(R₂)—(R₃)_(n)—Ywherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStreptococcus pneumoniae, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order, among others.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. FabK polypeptide fragments may be“free-standing” or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region ina single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Streptococcus pneumoniae, are also preferred.Further preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions, andsubstrate binding regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

A preferred embodiment of the invention is a protein wherein the aminoacid residue at position 318 is Ala; however, the residue at amino acidnumber 318 can be any one of the naturally occurring amino acid residuesexcept for threonine.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodeFabK polypeptides.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding FabK polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1], including a full lengthgene, or a variant thereof. This invention further provides that thisfull length gene is essential to the growth and/or survival of anorganism that possesses it, such as Streptococcus pneumoniae.

As a further aspect of the invention there are provided isolatedpolynucleotides encoding and/or expressing FabK polypeptides andpolynucleotides, particularly Streptococcus pneumoniae FabK polypeptidesand polynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically or clinically usefulpolynucleotides and polypeptides, and variants thereof, and compositionscomprising the same.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a FabK polypeptidehaving a deduced amino acid sequence of Table 1 [SEQ ID NO:2], andpolynucleotides closely related thereto, as well as variants thereof.

In another particularly preferred embodiment of the invention there is aFabK polypeptide from Streptococcus pneumoniae comprising or consistingof an amino acid sequence of Table 1 [SEQ ID NO:2], or a variantthereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding FabK polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in Table 1 [SEQ ID NO:1], typically alibrary of clones of chromosomal DNA of Streptococcus pneumoniae 0100993in E.coli, or some other suitable host, is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a sequencefragment of the invention. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent hybridizationconditions. By sequencing the individual clones thus identified byhybridization with sequencing primers designed from the originalpolypeptide or polynucleotide sequence, it is then possible to extendthe polynucleotide sequence in both directions to determine a fulllength gene sequence. Conveniently, such sequencing is performed, forexample, using denatured double-stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(See in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Direct genomic DNAsequencing may also be performed to obtain a full length gene sequence.Illustrative of the invention, each polynucleotide set out in Table 1[SEQ ID NO:1] was discovered in a DNA library derived from Streptococcuspneumoniae 0100993.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 975 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence that has at least 95% identity, even more preferably at least97–99% or exact identity to SEQ ID NO:1 over the entire length of SEQ IDNO:1; (b) a polynucleotide sequence encoding a polypeptide that has atleast 95% identity, even more preferably at least 97–99% or 100% exact,to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof; andisolating a full length gene and/or genomic clones comprising saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821–824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 975 set forth in SEQ ID NO:1 ofTable 1, both of that encode a FabK polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:X—(R₁)_(m)—(R₂)—(R₃)_(n)—Ywherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3′ end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5′ end nucleic acid residue is at the left,bound to R₁, and its 3′ end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, that can be adouble-stranded polynucleotide wherein the formula shows a first strandto which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Streptococcus pneumoniae, however, it may preferably be obtainedfrom other organisms of the same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms of thesame taxonomic family or order, among others.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae FabKhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may comprise coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingFabK variants, that have the amino acid sequence of FabK polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, modified, deleted and/oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesor activities of FabK polypeptide.

Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nucleicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO:1.

Further preferred embodiments of the invention are polynucleotides thatare at least 95% or 97% identical over their entire length to apolynucleotide encoding FabK polypeptide having an amino acid sequenceset out in Table 1 [SEQ ID NO:2], and polynucleotides that arecomplementary to such polynucleotides. Most highly preferred arepolynucleotides that comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to FabK polynucleotide sequences, such as thosepolynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. A specific exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycomprising a complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding FabK and to isolate cDNAand genomic clones of other genes that have a high identity,particularly high sequence identity, to a FabK gene. It is preferredthat these probes be used in the diagnostic embodiments provided herein.Such probes generally will comprise at least 15 nucleotide residues orbase pairs. Preferably, such probes will have at least 30 nucleotideresidues or base pairs and may have at least 50 nucleotide residues orbase pairs. Particularly preferred probes will have at least 20nucleotide residues or base pairs and will have less than 30 nucleotideresidues or base pairs.

A coding region of a FabK gene may be isolated by screening using a DNAsequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine to which members ofthe library the probe hybridizes.

There are several methods available and well known to those skilled inthe art to obtain full length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998–9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery ofdiagnostics for diseases, particularly human diseases, as furtherdiscussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue or bodily fluids. It isrecognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to a mature polypeptide (when amature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from a mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

As will be recognized, the entire polypeptide encoded by an open readingframe is often not required for activity. Accordingly, it has becomeroutine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol is known in the art and available from Promega (Madison,Wis.)). The digested endpoints can be repaired (e.g., by ligation tosynthetic linkers) to the extent necessary to preserve an open readingframe. In this way, the nucleic acid of SEQ ID NO:1 readily providescontiguous fragments of SEQ ID NO:2 sufficient to provide an activity,such as an enzymatic, binding or antibody-inducing activity. Nucleicacid sequences encoding such fragments of SEQ ID NO:2 and variantsthereof, as described herein, are within the scope of the invention, asare polypeptides so encoded.

As is known in the art that portions of the N-terminal and/or C-terminalsequence of a protein can generally be removed without seriousconsequence to the function of the protein. The amount of sequence thatcan be removed is often quite substantial. The nucleic acid cutting anddeletion methods used for creating such deletion variants are now quiteroutine. Accordingly, any contiguous fragment of SEQ ID NO:2 whichretains at least 20%, preferably at least 50%, of an activity of thepolypeptide encoded by the gene for SEQ ID NO:2 is within the invention,as are corresponding fragment which are 70%, 80%, 90%, 95%,97%, 98% or99% identical to such contiguous fragments. In one embodiment, thecontiguous fragment comprises at least 70% of the amino acid residues ofSEQ ID NO:2, preferably at least 80%, 90% or 95% of the residues.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (that may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci E. coli,streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcuspneumoniae; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Cajndida albicans and Aspergillus;insect cells such as cells of Drosophila S2 and Spodoptera Sf9; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowesmelanoma cells; and plant cells, such as cells of a gymnosperm orangiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of FabK polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof FabK polynucleotides and/or polypeptides in a eukaryote, particularlya mammal, and especially a human, will provide a diagnostic method fordiagnosis of disease or staging of disease. Eukaryotes, particularlymammals, and especially humans, particularly those infected or suspectedto be infected with an organism comprising the FabK gene or protein, maybe detected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled FabK polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397–4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingFabK nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

In another aspect, the present invention relates to a diagnostic kitthat comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2. Itwill be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferably, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from or is associated withexpression, particularly under-expression, over-expression or alteredexpression, of a polynucleotide of the invention. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing a firstphenotype but not in any organisms possessing a second phenotype, thenthe mutation is likely to be a causative or associated agent of thefirst phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, among other things. For example,RT-PCR can be used to detect mutations in the RNA. It is particularlypreferred to use RT-PCR in conjunction with automated detection systems,such as, for example, GeneScan. RNA, cDNA or genomic DNA may also beused for the same purpose. As a further example, PCR primerscomplementary to a polynucleotide encoding FabK polypeptide can be usedto identify and analyze mutations. The invention further provides theseprimers with 1, 2, 3 or 4 nucleotides removed from the 5′ and/or the 3′end. These primers may be used for, among other things, amplifying FabKDNA and/or RNA isolated from a sample derived from an individual, suchas a bodily material. The primers may be used to amplify apolynucleotide isolated from an infected individual, such that thepolynucleotide may then be subject to various techniques for elucidationof the polynucleotide sequence. In this way, mutations in thepolynucleotide sequence may be detected and used to diagnose and/orprognose the infection or its stage or course, or to serotype and/orclassify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, a polynucleotide having asequence set forth in Table 1 [SEQ ID NO:1]. Moreover, increased ordecreased expression of a FabK polynucleotide can be used as adiagnostic, and be measured using any one of the methods well known inthe art for the quantitation of polynucleotides, such as, for example,amplification, PCR, RT-PCR, RNase protection, Northern blotting,spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of FabK polypeptide compared to normal controltissue samples (usually showing no such expression) may be used todetect the presence of an infection, for example. Assay techniques thatcan be used to determine levels of a FabK polypeptide, in a samplederived from a host, such as a bodily material, are well-known to thoseof skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis, antibody sandwichassays, antibody detection and ELISA assays.

In a preferred embodiment of the invention, FabK polypeptide orpolynucleotide may be used in assays described herein to detectinfection by strains of Streptococcus pneumoniae that do not possess athreonine at amino acid position 318, such as Streptococcus pneumoniae0100993, the deposited strain. One may also type bacteria using thesepolypeptides or polynucleotides as described elsewhere herein.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

“Disease(s)” means any disease caused by or associated with infection byan organism, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

“Host cell(s)” is a cell that has been introduced (e.g., transformed ortransfected) or is capable of introduction (e.g., transformation ortransfection) by an exogenous polynucleotide sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403–410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403–410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443–453 (1970)

-   Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.    Natl. Acad. Sci. USA. 89:10915–10919 (1992)-   Gap Penalty: 12-   Gap Length Penalty: 4-   A program useful with these parameters is publicly available as the    “gap” program from Genetics Computer Group, Madison Wis. The    aforementioned parameters are the default parameters for peptide    comparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443–453 (1970)

-   Comparison matrix: matches=+10, mismatch=0-   Gap Penalty: 50-   Gap Length Penalty: 3-   Available as: The “gap” program from Genetics Computer Group,    Madison Wis. These are the default parameters for nucleic acid    comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:n _(n) ≦x _(n)−(x_(n) ·y),wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:n _(a) ≦x _(a)—(x _(a) ·y),wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

“Individual(s)” means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Organism(s)” means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii, Helicobacter pylori and Chlamydia trachomitis,(ii) an archaeon, including but not limited to Archaebacter, and (iii) aunicellular or filamentous eukaryote, including but not limited to, aprotozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces,or Candida, and a member of the species Saccharomyces ceriviseae,Kluveromyces lactis, or Candida albicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1–12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626–646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48–62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5–10, 1–5, 1–3,1–2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Cloning of Streptococcus pneumoniae FabK

The S. pneumoniae fabK, enoyl-ACP reductase gene was PCR amplified fromS. pneumoniae strain 0100993. The forward and reverse primer sequenceswere

5′ AGGTTGGAGGCCATATGAAAACGCGTATT 3′ (SEQ ID NO:3) and

5′ GGCGGATCCTTAGTCATTTCTTACAACTC 3′ (SEQ ID NO:4), respectively. An NdeIsite was integrated into the forward primer and a BamHI site into thereverse primer for cloning into pET24b(+). The PCR product was digestedwith the restriction endonucleases NdeI and BamHI and then ligated intopET24b(+), (also digested with NdeI and BamHI). The resulting plasmidwas transformed into sub-cloning efficiency DH5-alpha cells. Thesequence of the pET24bSpfabK expression construct was confirmed by DNAsequencing and the plasmid was transformed into electrocompetent BL21(DE3) cells harboring the tRNA vector pRR692.

Intact FabK is expressed as 25% total cell protein of which 80% issoluble when induced with 0.1 mM IPTG at 37° C. for three hours.

Example 2 Purification of S.pneumoniae FabK

One liter of cells containing the FabK expression construct were grownto an OD600 of 0.6. Expression was induced with 0.1 mM IPTG and thecells were grown for a further 3 h and then harvested. The cell pelletwas resuspended in 10 ml 50 mM Tris pH7.5, 1 mM PMSF, 1 mM Benzamidine,1 mM DTT (buffer A) and lysed by sonication. Cell debris was removed bycentrifugation. The supernatant was loaded onto a Hi-load Q (16/10)column (Pharmacia) equilibrated in buffer A. Protein was eluted over a200 ml gradient of 0–100% buffer B, where buffer B is buffer A+1 M KCl.Fractions containing FabK were identified by their absorbance at A460and by their FabK activity and pooled.

1.5 M ammonium sulphate was added to the pooled fractions and these werethen loaded onto a Hi-load Phenyl sepharose (16/10) column (Pharmacia)equilibrated in 50 mM Tris pH 7.5, 1 mM PMSF, 1 mM Benzamidine, 1 mMDTT, 1.5 M ammonium sulphate. Proteins were eluted with a gradient ofammonium sulphate (1.5 to 0 M) over 200 ml. Fractions containing FabKwere identified as above and pooled. The pooled fractions were bufferexchanged into 100 mM Tris, pH 7.5, 2 mM DTT and glycerol was then addedto 50%. The protein was stored at −20° C. It is preferred that theenzyme be stored with NH₄Cl, which has been found to stabilize theenzyme.

Example 3 FabK Characterization

The identity of the protein was confirmed by N-terminal sequencing andMALDI mass spectrometry. The optical spectrum of the protein wascharacteristic of flavoproteins, showing an absorbance in the 450 nmregion. The FAD cofactor was removed by denaturation of the protein andquantified. The ratio of FAD:protein was shown to be approximately 1:1.

Example 4 Assaying the Activity of FabK

FabK catalyses the reduction of enoyl-ACPs with the concommitantoxidation of NADH. Crotonoyl-ACP can be prepared as described below. Thereduction of crotonoyl-ACP to butyryl-ACP can be monitored by followingthe change in absorbance at 340 nm as NADH is oxidised.

Assays were carried out in Costar 3696 half-area plates in a final assayvolume of 150 μl on a Spectramax platereader. Substrates, NADH andcrotonoyl ACP, were incubated with FabK enzyme in 100 mMN-[2-acetamido]-2 iminodiacetic acid (ADA), pH 6.5, 100 mM NH₄Cl, 4%glycerol at 30° C. and the reaction monitored at 340 nm. This assayingcan also be performed using crotonyl CoA, NADPH or an NADH analogue as asubstrate.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1. An isolated polynucleotide comprising a nucleotide sequence encodingthe polypeptide of SEQ ID NO:
 2. 2. The isolated polynucleotide of claim1, further comprising a transcriptional regulatory sequence operablylinked to said polynucleotide sequence.
 3. The isolated polynucleotideof claim 1, wherein said polypeptide is fused to a heterologous aminoacid sequence.
 4. An isolated vector comprising the isolatedpolynucleotide sequence of claim
 1. 5. An isolated host cell comprisingthe vector of claim
 4. 6. The host cell of claim 5, wherein said hostcell is prokaryotic or eukaryotic.
 7. The host cell of claim 6, which isa prokaryotic cell.
 8. The host cell of claim 6, which is a eukaryoticcell.
 9. The isolated polynucleotide of claim 1, consisting of anucleotide sequence encoding the polypeptide of SEQ ID NO:
 2. 10. Theisolated polynucleotide of claim 1, comprising the polynucleotide of SEQID NO:
 1. 11. The isolated polynucleotide of claim 10, consisting of thepolynucleotide of SEQ ID NO:
 1. 12. The prokaryotic cell of claim 7,which is an isolated bacterial cell.
 13. The eukaryotic cell of claim 8,which is an isolated mammalian cell.